The machinery responsible for making proteins (e.g. ribosomal RNA, ribosomal proteins, translation factors, and tRNAs) is central to growth and development of all organisms. The control of its synthesis has been a central issue in molecular microbiolgy for almost 60 years. It had become clear in previous project periods that our work was central not only for understanding ribosome synthesis but also for understanding the mechanism of transcription initiation in general. What has become apparent in the last few years is that the systems that regulate rRNA promoters also directly regulate a wide variety of other promoters as well, many of which were not anticipated because their connection to the protein synthetic apparatus was not apparent. However, we now know that the molecules that regulate rRNA promoters are utilized throughout the bacterial kingdom, they play crucial roles in virulence and infectious disease, and they contribute to genome maintenance as well as to transcription. In the next project period, we will (i) continue our studies on the DNA-RNAP interactions that contribute to the transition from a closed to an open complex in the promoter mechanism, (ii) explore the role of specific promoter regions in defining the transcription start site, (iii) continue our examination of the control of transcription by ppGpp and DksA by defining their binding sites on RNA polymerase and determining their mechanism of action, (iv) expand our analysis of control by these molecules to additional promoters in order to understand what differentiates positively from negatively regulated targets, (v) continue studying the mechanism of action ofthe RNA polymerase assembly factor CrI, and (vi) continue studies on our recent discovery that E. coli makes a structure analogous to the eukaryotic nucleolus. We will expand these studies to explore other potential long-range interactions between distant parts of the bacterial chromosome.